The LHD has a long history of investigating patients with abnormalities of phagocytic cell function. These studies include the delineation of clinical, functional, and molecular defects of patients with neutrophil specific granule deficiency, chronic granulomatous disease (CGD), leukocyte adhesion deficiency (LAD), the syndrome of hyperimmunoglobulinE and recurrent infections (Jobs Syndrome) and IRAK4 deficiency. Large cohorts of these patients have been recruited over the years and represent a unique national resource for biomedical research at the NIH. Currently we follow over 150 patients with CGD, about 40 patients with Jobs syndrome, and 30 patients with other phagocyte dysfunction syndromes, including LAD, cyclic neutropenia, neutrophil specific granule deficiency, Chediak-Higashi syndrome, IRAK4 deficiency and NEMO deficiency. We now have EB virus transformed B cells from most of our patients and we have been pleased to share these B cell lines with other intramural or extramural colleagues. We continue to monitor and expand these cohorts of patients who serve as models for long term studies of the clinical consequences of the immune dysfunction in humans. In 2009 we continued our studies of the importance of lactoferrin in protecting against Aspergillus fumigatus infection, the most common infectious cause of mortality in CGD patients today. We found that while CGD PMN are unable to kill Aspergillus hyphae, their ability to arrest the growth of conidia was identical to that of normal PMN showing a role for nonoxidative mechanisms in host defenses against this organism. We then showed that the neutrophil secretory product, lactoferrin, inhibits conidia germination by sequestering iron, a critical growth factor. We have continued studying the growth inhibitory properties of iron chelating drugs against Aspergillus fumigatus, alone and in combination with first line antibiotics such as amphotericin B, itraconazole, and fluconazole and demonstrated antifungal synergy in some combinations. Combinatorial fungal effects against A. fumigatus conidia were synergistic for ketoconazole with either ciclopirox or deferiprone, lactoferrin with amphotericin B, and fluconazole with deferiprone. Desferoxamine enhanced fungal growth. With the biostatistics branch, we developed a new statistical method for the analysis of synergy. Further studies have demonstrated significant antifungal activity of some iron chelators against other fungi (Cryptococcus neoformans, Candida albicans) suggesting an important new avenue for prevention and treatment of fungal infections common to immunocompromised patients. We are actively working with June Kwon-Chung (LID) to test whether these drugs work in vivo in murine aspergillosis. (Kol Zarember, 20% effort). In 2009 we completed our gene sequencing data and phenotype correlates in 259 patients with chronic granulomatous disease (CGD) representing 221 distinct families, a cohort large enough to provide sufficent statistical power to compare specific CGD subtypes. We have also analyzed 40 X-linked heterozygous carriers in 34 families and 9 mothers who have children with spontaneous mutaions in gp91phox, but do not display heterozygosity. This cohort included unreported mutations in gp91phox (44 mutations), in p67phos (9 mutations), and in p22phox (1 mutation). Missense and frameshift mutations in gp91phox are randomly distributed throughout the protein and are generally family-specific. However, nonsense mutations are more discrete, preferentially localized to CpG dinucleotides "hotspots" that are common to as many as 9 separate families. Furthermore, 4 of the patients with spontaneous CGD exhibit de novo mutations at CpG dinucleotides. Analysis of gp91phox mutations indicates that missense mutations with the carboxyl terminal FAD and NADPH nucleotide binding domains almost completely abrogate the activity of the oxidase, but often do not alter the expression and/or stability of gp91phox. Muations in the amino terminal transmembrane region of gp91phox, particularly the extracellular domains, are more permissive, retaining some activity of the oxidase, but generally with diminished expression and/or stability of the enzyme. Survival analysis of the major CGD genotypes indicates that patients with missense mutations in gp91phox and patients with mutations in p47phox exhibit survival significantly better than patients with nonsense, frameshift, RNA processing, and deletion mutations in gp91phox mutations. Moreover, patients with missense mutations in gp91phox are not significantly different from the survival curve of the general US population. These data indicate that CGD phenotype can be used to predict NADPH oxidase function and survival in patients with CGD. In other related studies in a few gp91phox deficient CGD patients it appears that patients with missense mutations are responsive to interferon gamma in terms of superoxide production suggesting mutational analysis may predict interferon gamma responsiveness. Over the past year, the laboratory has expanded its study of Granulibacter bethesdensis, a recently described bacterial pathogen of CGD patients. Granulibacter is remarkably hypostimulatory of the innate immune system, both in terms of weak activation of the NADPH oxidase and poor stimulation of cytokine secretion. Ongoing efforts to purify and characterize what apperas to be an atypical lipolysaccharide (LPS) of this organism will expand in coming months. We have isolated a fraction that appears to represent the Lipid A of this organism that has strongly stimulatory activity in the limulus LPS test but fails to activate human PMN in keeping with our findings that this microbe may avoid host defenses by using molecular stealth. We have also found that G. bethesdensis is remarkably resistant to complement and antimicrobial peptides. In collaboration with LCID, we are evaluating internalization and killing of Granulibacter by normal and CGD leukocytes. Further studies examining the transcriptional responses of the pathogen to attachk by the host and thost cells in response to the pathogen are underway. (Zarember 50% effort). In 2009 we studied the role of tryptophan metabolism in CGD. Mouse CGD models were recently implicated to have defective tryptophan catabolism as a major regulator of inflammation in CGD. We attempted to validate these findings in human clinical samples and discovered that unlike CGD mice, human CGD patients do not display this defect in tryptophan catabolism. (Zarember, 10% effort). In addition to these projects, in 2009 we have written a clinical protocol that will enroll patients to study the development of atherosclerotic disease in patients with immune system disorders. Atherosclerosis, the major cause of heart disease, is thought to relate to dysregulated inflammation. in the cardiac blood vessels and possibly results from over production of reactive oxygen species (ROS). We hypothesize that CGD patients, who have absent production of reactive oxygen species by their phagocytes may be protected from developing atherosclerosis. The primary endpoint of this study is to determine the prevalence of atherosclerosis in these and other patients with in-born disorders of immune function. The primary endpoint will be assessed using imaging techniques to measure coronary artery calcium scores and the presence of absence of soft plaque. Secondary endpoints include physiologic markers such as blood pressuer as well as circulating biomakers associated with heart disease. We expect to enroll patients into thei protocol in the fall of 2009. (Soule 15 % effort).